The technical field of this invention is cell culturing and, in particular, methods for separating malignant cells from biopsy samples or other clinical specimens.
The culturing of cancerous or otherwise abnormal cells has become an important tool of clinicians and researchers combating the diseases which such cells manifest. Because cancerous cells of identical histopathological type show a wide range of responsiveness to particular drug therapies among individual patients, predictive techniques, similar to the culture and sensitivity assays used for the management of microbial infections, have become valuable techniques for selecting effective chemotherapy in individual cases. Quick and effective culturing techniques are essential to such predictive assays.
Similarly, the culturing of cells from biopsy samples has also become important in establishing cell lines. Tumor registries provide standardized tumor cell lines, against which new drugs and other agents can be tested to screen for more effective modalities of treatment. Thus, effective techniques for separating malignant cells from clinical specimens are also essential to the establishment of primary cultures which can be expanded into cell lines for screening purposes.
Early predictive assay techniques involved the cloning of single cell suspensions from biopsy specimens in soft agar after brief exposure to particular anti-cancer drugs. These agar culture techniques often suffered from low plating efficiency and poor correlation with actual treatment results. See Salmon et al., "Quantitation Of Differential Sensitivity Of Human Stem Cells To Anti-Cancer Drugs," Vol. 298 New England Journal of Medicine, pp. 1321-1327 (1978); Buick et al., "Development Of An Agar-Methyl Cellulose Clonogenic Assay For Cells In Transition Cell Carcinoma Of The Human Bladder," Vol. 39 Cancer Research, pp. 5051-5056 (1979) and Von Hoff et al., "Direct Cloning Of Human Malignant Melanoma In Soft Agar Culture," Vol. 50 Cancer, pp. 695-701 (1982), for further details on agar culture techniques.
Another predictive system which has been proposed for chemotherapy studies involves the growth of cancer cells in vessels fed by a matrix of synthetic, hollow fiber capillaries. In such systems, single cell suspensions from biopsy samples were again used to seed the artificial capillary vessel. See, for example, Quartles et al., "Hemodialysis-Matrix Profusion Culture System: A New Technique For Studying Chemotherapeutic Activity In Tumor Cells," Vol. 16 In Vitro, p. 246 (1980). For a review of capillary cultures, generally, see Schratter, "Cell Culture With Synthetic Capillaries," Vol. XIV Methods In Cell Biology, pp. 95-103 (1976).
In U.S. Pat. No. 4,559,290, the present applicant has disclosed a simple, sensitive assay technique in which cancerous or otherwise abnormal cells can be cultured and their response to chemotherapeutic agents evaluated by measuring the retention of fluorescein or similar label by living cell membranes. The cultured cells are allowed to accumulate fluorescein by contact with a fluorogenic substrate, typically a nonpolar ester of fluorescein and an aliphatic acid, introduced into the cell culture. The fluorogenic substrate penetrates the cell membranes where it is enzymatically hydrolyzed, liberating fluorescein and staining the cell brightly fluorescent under blue light. Since fluorescein, a negatively charged molecule, does not diffuse readily across the cytoplasmic membrane of normal cells, the process causes intracellular accumulation of fluorescein in living cells. However, when a dead cell is treated with a fluorogenic substrate, no intracellular accumulation of fluorescein is observed.
One of the problems which has plagued clinicians and researchers in conducting chemotherapeutic assays and the like is the need to separate from a biopsy sample those cells which are responsible for the malignancy and then to culture such cells in a manner that mimics in vivo conditions. The conventional techniques for preparing cell lines involve the complete dissociation of the tumor specimens into single cell suspensions Unfortunately, such suspensions often contain a large number of normal cell which are very difficult to separate from tumor cells.
Moreover, when single cell suspensions are used for assay purposes, or even when cell lines are grown from such single cells, tests conducted on such cultures often are poorly correlated with actual results in patient treatment. The destruction of the primary tissue architecture has been shown to affect not only cellular ability to survive under culture conditions but drug sensitivity to anticancer agents as well. See, generally, Miller, B. E. et al. "Interactions between tumor subpopulations affecting their sensitivity to the antineoplastic agents cyclophosphamide and methotrexate." Vol. 41 Cancer Res., pp. 4378-4381 (1981) and Rasey, J. S. and Nelson, N. J. "Response of an in vivo-in vitro tumor to X-rays and cytotoxic drugs: effect of tumor disaggregation method on cell survival." Vol. 41, Br. J. Cancer Suppl. IV: pp. 217-221 (1980).
Thus, there exists a need for better techniques for preparing biopsy samples for use in predictive assays. A simple, effective way of separating viable malignant cells, substantially free of extraneous (i.e., non-malignant) cells would satisfy a long-felt need in the field.